Although human germ cell formation and differentiation undoubtedly shares similarity with that of the mouse, it is also clear that the genetic requirements for human germ cell development differ substantially from those of mice. This is most-plainly illustrated by contrasting the roles of the sex chromosomes in germ cell development in humans and mice. Several Y chromosome loci that are required for germ cell development in men are absent in mice. Similarly, in the case of the X chromosome, women require 2 X chromosomes for oocyte development, whereas, mice are fertile with just a single X chromosome. Because of differences such as these, we explored whether ES cells might provide a much sought after human-genome based system with which to manipulate the genetics and epigenetics of human germ cell specification and differentiation in vitro. Our preliminary data indicates that germ cells can be specified and differentiated from human ES cells. Thus, we hypothesize that the human ES cell system can be used to specifically probe the genetics of human germ cell formation and differentiation. Moreover, we hypothesize that the downregulation of key genes that map to a region of the Y chromosome that is commonly deleted in infertile men, the AZFc region will abolish the ability to form and/or differentiate human male germ cells in vitro and in vivo in a transplant system. To explore this hypothesis, we propose to: 1) Characterize further the ability of NIH-approved human ES cell lines to contribute to the germ cell lineage in vitro and in vivo. For this purpose, we will use human ES cell lines H1, H14 and HSF1 (NIH codes: WA01, WA14 and UC01). 2) Silence the Y chromosome genes that map to the AZFc deletion interval and assess germ cell formation in vitro. 3) Silence the Y chromosome genes that map to the AZFc deletion interval and assess the ability of germ cells to colonize primate spermatogenic tubules and subsequently differentiate. This work aims to develop a robust system to specifically probe the genetics of human germ cell development. As such, this research is significant to our understanding of basic germ cell development in humans and has the direct potential to increase knowledge of germ cell defects in the 10-15% of couples who are infertile.